The long-term goal of this work is to understand, in molecular detail, the regulation and catalytic mechanism of serine recombinases. These site-specific DNA recombinases are widespread in prokaryotes and perform a wide variety of genetic manipulations, often associated with the spread and/or stability of resistance genes. They are also useful tools for manipulating the genomes of other organisms. However, they are not as well understood as the other family of site-specific recombinases, the tyrosine or ? integrase family (which includes Cre and Flp). This work will focus on the resolvase subfamily of serine recombinases, using Sin as a prototype. Sin is encoded by many large multi-resistance plasmids of Staph. aureus, and is thought to promote their stable maintenance by resolving replicon dimers into monomers. A remarkable feature of the Serine resolvases is their regulation: the WT enzymes will catalyze intrabut not intermolecular recombination, can sense the relative orientation of their sites, and can exchange strands directionally despite the fact that there is no net release of chemical bond energy. This key to this regulation is that they are only active within a large, intertwined complex called the "synaptosome." Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a "topological filter." Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. How the regulatory complex activates the dimers bound to the paired crossover sites is unknown. The mechanism used by these enzymes to catalyze the breakage and reunion of DNA strands is also poorly understood. It is known that they form phosphoserine intermediates, but the structures available to date do not show a fully assembled active site, making it difficult to interpret the chemical roles of conserved residues. Understanding how the synaptic complexes of the serine resolvases regulate the chemical and mechanical steps of recombination should be applicable to other serine recombinases: even for those that require a different regulatory apparatus, the molecular details of the inactive-to-active transition is likely to be analogous. The approach taken in this study is a combination of in vitro biochemical studies and x-ray crystallography, in close collaboration with a group that has expertise with genetics, biochemistry, and DNA topology. PUBLIC HEALTH RELEVANCE This work probes the mechanism and regulation of a DNA recombination reaction that is important for stable maintenance of many drug resistance-carrying plasmids found in Staphylococcus aureus, a common cause of opportunistic infections. Closely related recombination systems found in other bacteria also aid in the maintenance and/or mobility of resistance genes. This work will lead to a better understanding of genetic transactions in bacteria, enhancing the predictive power of sequence databases and leading toward ways to control resistance genes in bacteria.